Selectively bred rodent models for studying the etiology of type 2 diabetes: Goto-Kakizaki rats and Oikawa-Nagao mice

Abstract

Type 2 diabetes (T2D) is a polygenic disease and studies to understand the etiology of the disease have required selectively bred animal models with polygenic background. In this review, we present two models; the Goto-Kakizaki (GK) rat and the Oikawa-Nagao Diabetes-Prone (ON-DP) and Diabetes-Resistant (ON-DR) mouse. The GK rat was developed by continuous selective breeding for glucose tolerance from the outbred Wistar rat around 50 years ago. The main cause of spontaneous hyperglycemia in this model is insulin secretion deficiency from pancreatic β-cells and mild insulin resistance in insulin target organs. A disadvantage of the GK rat is that environmental factors have not been considered in the selective breeding. Hence, the GK rat may not be suitable for elucidating predisposition to diabetes under certain environmental conditions, such as a high-fat diet. Therefore, we recently established two mouse lines with different susceptibilities to diet-induced diabetes, which are prone and resistant to the development of diabetes, designated as the ON-DP and ON-DR mouse, respectively. The two ON mouse lines were established by continuous selective breeding for inferior and superior glucose tolerance after high-fat diet feeding in hybrid mice of three inbred strains. Studies of phenotypic differences between ON-DP and ON-DR mice and their underlying molecular mechanisms will shed light on predisposing factors for the development of T2D in the modern obesogenic environment. This review summarizes the background and the phenotypic differences and similarities of GK rats and ON mice and highlights the advantages of using selectively bred rodent models in diabetes research.

Introduction

Type 2 diabetes (T2D) is a disease of chronic hyperglycemia characterized by the two major pathogenic factors, insulin resistance and defective insulin secretion. Since obesity is a strong risk factor of insulin resistance and for the further development of T2D, most of animal models of T2D are obese [1, 2]. Among them, monogenic models defective in leptin signaling (e.g., db/db mice and Zucker diabetic fatty rats) are widely used for T2D research [2]. However, the incidence of T2D in humans is not explained by a monogenic mutation for obesity and a failure of β-cell adaptation to the increased demand of insulin in the face of insulin resistance is also indispensable for the development of T2D. From these points, polygenic models, i.e., selectively bred animal models, are considered more suitable to mimic the etiology of T2D in humans. Selective breeding is used to develop animal and plant strains with specific phenotypic traits. In the process of selection, genetic variants responsible for desirable traits can accumulate in subsequent generations. Selectively bred animal models may also be useful for elucidating genotype-phenotype interactions in complex traits involving multiple genes, such as glucose tolerance. So far, two selective breeding studies were performed solely for glucose tolerance, evaluated by an oral glucose tolerance test (OGTT), as a desirable trait to establish polygenic animal model of T2D. In the 1970s, Goto-Kakizaki (GK) rats were established by repetitive selective breeding for spontaneous glucose tolerance from the outbred stock of Wistar rats. Recently, we have established another rodent model of T2D, Oikawa-Nagao (ON) mice, by selective breeding for different susceptibilities to diet-induced diabetes. ON mice were developed by repeated selection for both inferior and superior glucose tolerance after high-fat diet (HFD) feeding, for Diabetes-Prone (ON-DP) and Diabetes-Resistant (ON-DR) lines, respectively, in hybrid mice of three inbred strains. Employing HFD as a dietary factor in the ON mouse breeding strategy may play a key role in deciphering predisposing factors for the development of T2D in the modern obesogenic environment. In this review, we first outline the development of GK rats and ON mice and their glucose tolerance phenotype as the desirable trait. These topics have already been summarized in our previous review [3] but are reiterated here to promote understanding of the following subjects. Then, we summarize pathogenesis of glucose intolerance, in particular impaired insulin secretion, of these models and its relevance to the pathogenesis of T2D in humans, with some recent updates. Finally, we compare the phenotypic features of GK rats and ON mice to highlight the potential advantages of using the selectively bred rodent models in diabetes research.

Goto-Kakizaki (GK) Rats

Background

With the aim to investigate if diabetes is an inheritable disease, Yoshio Goto and colleagues established the GK rat. Nine males and nine females with higher post-challenge blood glucose levels were selected as breeding stock from the initially performed an OGTT (2 g/kg) on 130 male and 81 female Wistar rats [4]. Selection and breeding of the descendants with higher blood glucose levels in the OGTT was continued over multiple generations. The sum of blood glucose values during the OGTT (SUM_BG; the sum of blood glucose levels measured at fasting and at 30, 60, 90 and 120 min) was increased gradually as the generations proceeded. The distribution of SUM_BG was completely separated from that of the control Wistar rat at the 9th generation and finally reached a plateau at the 15th generation [5]. Breeding pairs of GK rats from the original colony (the Sendai colony; GK/Sen) have been distributed worldwide for more than 40 years and local colonies have been established around the world [6-10]. Nowadays, GK rats are available not only from various local colonies but also from some commercial breeders in Japan, USA and Europe. Most of the colonies are maintained as inbred lines through sister-brother mating [11, 12].

Phenotype

The glucose intolerance in GK rats is suggested to be primarily caused by impaired insulin secretion due to defective β-cell function [13, 14]. The blood glucose levels and other characteristics (e.g., islet morphology and function) of GK rats vary among the colonies [12, 15]. These differences may be influenced by local breeding environment and/or genetic variation [11, 15]. For more information about the characteristics of each colony, see e.g., reviews by Portha et al. [15] and Östenson et al. [12, 16]. As example, our measurement in the adult GK rats of the Lund University colony (GK/LU rats) show non-fasting blood glucose levels of 20–25 mM, which is around three times higher than control Wistar rats [3]. The blood glucose level of GK/LU rats increased rapidly up to 10 weeks of age, reached a maximum at ~15 weeks of age, and declined slightly from ~25 weeks of age. The acute development of hyperglycemia after weaning in GK/LU rats is similar to the other colonies, and has been explained primarily by the acquired hepatic insulin resistance [14, 17] and secondarily by the insufficient β-cell compensation for the insulin resistance [18-20]. There was no significant difference in plasma insulin levels, but body weight is ~20% lower in GK/LU rats than Wistar rats of the same age.

A large proportion of the pancreatic islets of adult GK/LU rats are characterized by decreased β-cell number, increased fibrosis and a random distribution of α- and δ-cells throughout the core. Moreover, the islets have an irregular shape with ill-defined borders and fibrous strands traversing the islet, so-called “starfish-shaped islets” (Fig. 1) [14, 21, 22]. The development of fibrosis in the islets is suggested to be the result of chronic inflammation [23]. Adult GK rat islets are infiltrated with macrophages [24] and show increased gene expression levels of proinflammatory cytokines and chemokines [25]. We and others have demonstrated decreased insulin content and a reduced β-cell mass in islets of GK/LU rats [3, 26]. This is consistent with a decreased β-cell population and/or degranulation of the β-cells shown in adult GK rat islets from other colonies [14, 21, 27]. However, this is not always true as there are examples where neither β-cell mass nor islet insulin content were decreased even in adult GK rats [15, 16]. These findings suggest that the primary defect in the GK rat is β-cell dysfunction.

Fig. 1

Shapes of pancreatic islets of Goto-Kakizaki (GK) and Wistar rats.

Appearances of pancreatic islets from 12-week-old Goto-Kakizaki (GK/LU, A) and Wistar rats (B). The images were taken using a confocal microscope (×10).

Pathogenesis

The sequence of events leading to insulin secretion in response to glucose in β-cells is termed “stimulus-secretion coupling”. During the process, glucose uptake and glycolysis lead to mitochondrial ATP production, closure of ATP-sensitive K⁺ (K_ATP) channels, membrane depolarization, opening of voltage-dependent Ca²⁺ (Ca_V) channels, and finally exocytosis of insulin granules and insulin release [28, 29]. Several defects are coupled to the stimulus-secretion coupling in GK rat β-cells. First, the GK rat islets have reduced expression of the glucose transporter 2 (GLUT2) [30, 31], which presumably have a negative impact on glucose uptake. Glycolysis has been suggested to be intact [6, 9, 32, 33], but the mitochondrial glycerol phosphate shuttle has been consistently reported to be impaired in GK rat β-cells [34-38]. These defects could account for the reported abnormalities in glucose-stimulated ATP production [33, 39] and downstream K_ATP channel closure [40] and cytoplasmic Ca²⁺ elevation [41, 42]. The ion channel properties of K_ATP and Ca_V channels seem to be intact as suggested by a series of patch-clamp experiments on GK rat β-cells [33, 40, 43].

Several indications have shown that the process of exocytosis of insulin granules involved in the final step of the stimulus-secretion coupling is impaired in GK rat β-cells. Reduced exocytosis in GK rat β-cells as compared to Wistar rat ones has been demonstrated by using patch-clamp in combination with capacitance measurements on single β-cells [43, 44]. Changes in membrane capacitance reflect exocytosis of insulin granules, which is initiated by the opening of Ca_V channels followed after the membrane depolarization. Initial exocytosis (increase in membrane capacitance) has been suggested to link to first phase insulin secretion. On the cellular level, first phase insulin secretion represents the exocytosis of previously docked granules [45, 46]. Hence, reduced first phase insulin secretion in GK rat islets [6] suggests a decreased number of docked granules at the plasma membrane in GK rat β cells. The molecular machinery of exocytosis requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which brings the granular membrane in close contact with the plasma membrane for bridging the membranes for fusion [29]. The SNARE complex components (e.g., STX1A, VAMP2, SNAP25 and STXBP1) are markedly reduced in the islets of GK rats [47-50], which at least partly explain the reduced exocytosis.

Glucose-stimulated insulin secretion (GSIS) can be augmented by incretins, glucagon-like peptide 1 (GLP-1) and gastric insulinotropic polypeptide (GIP), through stimulating β-cell electrical activity and enhancing Ca²⁺ signaling for accelerating the process of exocytosis [29, 51]. GLP-1 and GIP bind to stimulatory G protein (G_s)-coupled receptors (GLP1R and GIPR, respectively) and activate adenylyl cyclase (AC) to generate cAMP, the intracellular mediator [52]. A genetic variant in GK rats, within the Niddm1i locus, is associated with reduced cAMP levels in the β-cells, as described in the following section [53]. Both GLP1R and GIPR positive cells have been suggested to be decreased in the islets of GK rats [26]. However, expression levels of G_s, AC1 and AC3 are rather increased in GK rat β-cells [54]. Notably, two point mutations at the promoter region of AC3 are associated with the increased expression level of AC3 in GK rats [55]. Furthermore, cocaine- and amphetamine-regulated transcript (CART), which can increase intracellular cAMP through AC activation, is upregulated in GK rat β-cells [56]. Thus, it seems reasonable that cAMP inducers, e.g., forskolin, acetylcholine and GLP-1, can restore impaired glucose-induced insulin secretion in GK rat islets [18, 57, 58].

Implications for human T2D

Several studies have been performed to identify the genomic regions in GK rats corresponding to human T2D loci [53, 59, 60]. In a congenic strain of GK rats, one locus was linked to impaired β-cell function [59]. The region in focus, Niddm1i, is located on chromosome 1 in the GK rat genome. Interestingly, different segments within Niddm1i are suggested to be associated with distinct defects in glucose metabolism and exocytosis of insulin granules. The defect in exocytosis, including deteriorated granular docking, is linked to a 1.4 Mb genetic segment of Niddm1i, containing five known protein-coding genes (Pdcd4, Lysmd3, Shoc2, Adra2a and ENSRNOG00000036577). Among them, we demonstrated that overexpression of α2A-adrenargic receptor (α2A-AR), encoded by Adra2a, reduced granule docking at the plasma membrane and consequently decreased exocytosis [53]. The α2A-AR is coupled to an inhibitory G proteins (G_i) which can inhibit AC and decrease the cAMP production important for enhanced insulin secretion [61]. In humans, a single-nucleotide polymorphism in the ADRA2A gene was shown to be associated with increased risk of T2D. Furthermore, the polymorphism was linked to α2A-AR overexpression and defect insulin secretion in islets from T2D donors [53]. This is a good example where the findings in the GK-rat model was elongated to a human setting, showing the advantage of using GK-rats in studies for the etiology of diabetes.

The GK rat islets have a perturbed microRNA (miRNA) network that can contribute to the observed defects. miRNAs are small non-coding RNAs that can regulate protein expression, and several miRNAs are differentially expressed in T2D islets [62]. In our studies, we found that 24 miRNAs were upregulated and six downregulated in GK rat islets as compared to Wistar rat islets [63]. In line with a reduced exocytosis of insulin granules, some of the upregulated miRNAs may target genes involved in β-cell exocytosis. One of the miRNAs up-regulated in GK-rat islets is miR-335 which targets the exocytotic gene Stxbp1. Interestingly, miR-335 expression negatively correlated with insulin secretion in islets from human donors with impaired glucose tolerance (IGT), and the overexpression of miR-335 impaired insulin secretion by impairing priming of insulin granules [64]. Aside from targeting exocytotic genes, we have also demonstrated that other upregulated miRNAs (miR-130a/b and miR-152) in islets from GK rats are also up-regulated in islets from human donors with IGT and T2D. These miRNAs negatively regulates pyruvate dehydrogenase E1 alpha (PDHA1) and glucokinase (GCK), resulting in the alteration of intracellular ATP levels which affect ATP-requiring β-cell processes including glucose-induced insulin secretion, insulin biosynthesis and processing [65]. In addition to the perturbed miRNA network, the involvement of epigenetic transcriptional regulation should be taken into consideration in analyzing β-cell function in GK rats as epigenetic modification in islets from human T2D donors has been shown to be associated with reduced insulin secretion [66].

GK rats have also served as a useful animal model for diabetes research in various target organs as well as islets and β-cells. It corresponds to the clinical features of human T2D as a systemic disease. For instance, GK rats have a mild cardiac defect, characterized by diastolic dysfunction without ischemia [67-69], suggesting that GK rats are a good model to address the pathophysiology of diabetic macrovascular complication. Many researchers have also focused on the dysfunctions in nerves, kidneys and eyes as Goto and colleagues demonstrated the usefulness of GK rats as an animal model of microvascular complications [70-73]. Among them, GK rats are a well-studied model of diabetic nephropathy and neuropathy. An increase in albuminuria and a decline in renal function are observed in several GK rat colonies [74, 75], with thickening of the glomerular basement membrane is consistent across the colonies [70, 76]. As implications of neuropathy, motor nerve conduction velocity is reduced consistently in peripheral nerves of GK rats [77-79]. Insulin targeting organs (the liver, muscle and adipose tissue) are also potential research targets for elucidating the pathophysiology of insulin resistance, which plays a certain role in the development of hyperglycemia in GK rats. Furthermore, about half of the published studies using GK rats indexed in PubMed are intervention studies on hypoglycemic agents or other therapeutics [3]. Therefore, studies in GK rats, have led to fundamental breakthroughs not only in the etiology of T2D but also in therapeutic strategies for T2D and its complications.

Oikawa-Nagao (ON) Mice

Background

The selective breeding for two lines of ON mice, originally reported as Selectively bred Diet-induced Glucose intolerance-Prone/-Resistant (SDG-P/R) mice, was launched by Shinichi Oikawa in 2001 [80]. The original aim of the selective breeding was to examine whether HFD-induced glucose intolerance could be passed on to the next generations as a heritable trait. The selective breeding was performed using hybrid mice derived from three inbred strains, C57BL/6J, AKR/N and C3H/HeJ, to generate genetic diversity. This would play a crucial role in establishing the polygenic disease model animals. Initially, the hybrid mice were fed a HFD for 10 weeks and thereafter received an OGTT (glucose 2 g/kg). The mice with higher blood glucose levels at 120 min in the OGTT (BG_{120 min}) were selected for breeding. In the early generations of the breeding, we found that some mice maintained good glucose tolerance even after the HFD feeding. Since 2005, therefore, HFD fed mice showing both higher and lower BG_{120 min} were selected, and each was bred repetitively. As a result, two mouse lines with different susceptibilities (prone and resistant) to HFD-induced glucose intolerance were established [80]. During the selective breeding, sister-brother mating was avoided to maintain the genetic diversity and fertility after the HFD feeding. The selection of the prone and resistant lines was terminated when the distribution of BG_{120 min} values became distinctly different from each other [3]. At the end of the selective breeding (23rd and 22nd generation of SDG-P and SDG-R, respectively), SDG-P and -R lines were renamed to Oikawa-Nagao mouse Diabetes-Prone (ON mouse-DP^®, ON-DP) and Diabetes-Resistant (ON mouse-DR^®, ON-DR), respectively [3]. Both lines of ON mice are available from the Institute for Animal Reproduction (Kasumigaura, Japan) since 2022.

Phenotype

ON-DP mice under normal chow feeding show an IGT phenotype with modest hyperglycemia not in fasting but in post-challenge condition compared to ON-DR mice (Fig. 2A). After 5-week HFD feeding, male ON-DP mice develop to a diabetic phenotype with moderate fasting hyperglycemia and markedly higher post-challenge blood glucose levels in OGTT (Fig. 3A). HFD-fed female ON-DP mice have relatively lower post-challenge blood glucose levels compared to the HFD-fed male ON-DP mice, indicating that the females are less prone to HFD-induced hyperglycemia relative to the males. ON-DP mice gain more body weight under the HFD feeding and develop more severe insulin resistance compared to ON-DR mice [81]. The excessive weight gain of ON-DP mice can be explained by their hyperphagic behavior, as it was completely suppressed to the level of ON-DR mice by pair-feeding [82]. Pair-feeding with ON-DR mice attenuated the manifestation of insulin resistance and subsequent deterioration of glucose intolerance in ON-DP mice [82]. ON-DP mice also show higher inflammatory cytokine gene expression levels in the visceral fat and higher blood pressure after the HFD feeding compared to ON-DR mice [83].

Fig. 2

Glucose tolerance and pancreatic islet characteristics of Oikawa-Nagao Diabetes-Prone (ON-DP) and Diabetes-Resistant (ON-DR) mice at 5 weeks of age under normal chow feeding [81].

A: Blood glucose levels in OGTT (n = 5). B: Immunohistochemical images and morphometric analyses for α- and β-cells of pancreatic islet (n = 6). Double staining for insulin (brown) and glucagon (red). C and D: Glucose- and K⁺-induced insulin secretion (C, n = 9–10) and relative gene expression levels (D, n = 7–9) of isolated pancreatic islets. Gene expression levels were normalized to Gapdh, and the normalized expression levels of ON-DP mice were expressed as relative values to those of ON-DR mice. Male ON-DP and ON-DR mice at 5 weeks of age under normal chow feeding were analyzed. Mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. ON-DR mice (Student’s t-test).

Fig. 3

Glucose tolerance and pancreatic islet characteristics of Oikawa-Nagao Diabetes-Prone (ON-DP) and Diabetes-Resistant (ON-DR) mice at 10 weeks of age after high-fat diet feeding [81].

A: Blood glucose levels in OGTT (n = 6). B: Immunohistochemical images and morphometric analyses for α- and β-cells of pancreatic islet (n = 5–6). Double staining for insulin (brown) and glucagon (red). C and D: Glucose- and K⁺-induced insulin secretion (C, n = 8–9) and relative gene expression levels (D, n = 8) of isolated pancreatic islets. Gene expression levels were normalized to Gapdh, and the normalized expression levels of ON-DP mice were expressed as relative values to those of ON-DR mice. Male ON-DP and ON-DR mice at 10 weeks of age after 5-week high-fat diet feeding were analyzed. Mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. ON-DR mice (Student’s t-test).

Pathogenesis

ON-DP mice have modestly higher post-challenge blood glucose levels (Fig. 2A) and lower acute insulin response in OGTT performed at 5 weeks of age (under normal chow feeding) compared to ON-DR mice [81]. While the islet shape and β-cell mass of ON-DP mouse appeared to be normal (comparable to those of ON-DR mice) at 5 weeks of age (Fig. 2B), GSIS and K⁺-stimulated insulin secretion (KSIS) of ON-DP mouse islets was reduced compared to ON-DR (Fig. 2C) [81]. The lower insulin secretory response of ON-DP mouse islets was still observed after 5-week HFD feeding, but with a marked increase in β-cell mass, despite the reduced gene expression level of Pdx1 (Fig. 3B–D) [81]. Similar to the islets from GK rats and human donors with T2D, ON-DP mouse islets have reduced gene expression levels of Slc2a2 (Glut2) and exocytotic proteins (Stx1a and Snap25) (Fig. 2D) [81]. Considering that insulin content is rather increased in the ON-DP mouse islets [81], the stimulus-secretion coupling is impaired in ON-DP mouse β-cells and the defect exist likely at the level of glucose uptake and calcium-dependent exocytosis of insulin granules, or both. The gene expression pattern of ON-DP mouse islets (relative to ON-DR) was almost unchanged after 5-week HFD feeding (Fig. 3D), implying that the β-cell dysfunction is a hereditary character.

In relation to the hyperphagic behavior of ON-DP mice under HFD, we recently found a difference in secretory capacity of leptin, an anorexigenic hormone secreted by adipocytes, between ON-DP and ON-DR mice before the onset of obesity [82]. At 5 weeks of age, ON-DP mice have lower plasma leptin concentrations and adipose tissue leptin gene expression levels compared to body weight-matched ON-DR mice. Corresponding with the peripheral leptin status, ON-DP mice show lower anorexigenic leptin signaling in the hypothalamic arcuate nucleus, a center of feeding regulation, without apparent leptin resistance. An adipose tissue explant culture study revealed that ON-DP mouse adipose tissue has lower insulin-induced leptin production/secretion capacity than ON-DR. Moreover, ON-DP mice show higher DNA methylation levels at the leptin gene promoter region of adipocytes when compared with ON-DR mice. Accordingly, we consider that heritable lower leptin production capacity plays an important role in overfeeding-induced obesity and subsequent development of diabetes in ON-DP mice.

Implications for human T2D

As aforementioned, studies in ON mice suggest that β-cell function and feeding behavior are crucial determinants for the susceptibility to HFD-induced diabetes. Approaches to clarify the causes of β-cell dysfunction and hyperphagic behavior in ON-DP mice may therefore be worthwhile in elucidating the etiology of T2D, especially related to the modern obesogenic lifestyle.

For instance, ON-DP mouse islets show a higher gene expression level of Cd36 compared to ON-DR in contrast to the reduced gene expression levels of exocytotic proteins (Fig. 2D) [81]. We recently identified an increased protein expression level of CD36 in human islets from obese T2D donors [84]. CD36, also known as fatty acid translocase, facilitates cellular fatty acid influx at the plasma membrane [85]. Long-term exposure of islets to fatty acids diminishes glucose-induced insulin secretion [86, 87]. Accordingly, an increased level of CD36 is likely to contribute to β-cell dysfunction in T2D associated with obesity. We therefore investigated the effect of CD36 on β-cell function using INS-1 insulinoma cells [84]. As a result, CD36 overexpression in INS-1 cells led to decreased insulin secretion due to the reduced number of docked insulin granules, and the result was associated with the reduced expression levels of exocytotic proteins; SNAP25, VAMP2, and STXBP1. These findings are highly relevant to the fact that patients with T2D often have a loss of the first phase insulin secretion associated with the reduced expression levels of exocytotic proteins [45, 88]. Considering that the ON-DP mice have increased level of CD36 and reduced levels of the exocytotic genes, ON mice could serve as a good model for further investigation of the role of CD36 in islet function and the development of T2D.

In addition, female ON-DP mice with mild hyperglycemia have greater atherosclerotic lesion formation than ON-DR mice after an atherogenic diet feeding [89], suggesting that ON mice would be useful for studying the underlying mechanisms of diabetic macrovascular complications. Furthermore, we recently demonstrated that metformin treatment can attenuate the atherosclerotic lesion formation in ON-DP mice [90]. Considering that intensive glycemic control with metformin early in the clinical course of T2D reduced subsequent cardiovascular events in the long-term follow-up of the United Kingdom Prospective Diabetes Study [91], the mildly hyperglycemic ON-DP mice would be a suitable model for exploring intervention strategies for early-stage atherosclerosis in patients with IGT and T2D.

Comparison of GK Rats and ON Mice

We summarize the phenotypic features of GK rats and ON mice in Table 1 for comparison and would like to comment on some of the differences and similarities between these two T2D animal models. GK rats are known as an inbred rat strain, but genetic diversity has been observed between the colonies [11]. So far, ON mice are available as outbred strains, but sister-brother mating has been attempted in both lines after the end of the selective breeding. The differences between GK rats and ON mice in the onset and pathogenesis of diabetes are quite interesting. GK rats develop diabetes spontaneously and have a very short pre-diabetic window. On the other hand, ON-DP mice are in a pre-diabetic stage for longer periods of time under normal chow feeding. Thus, ON-DP mice may serve as a pre-diabetic animal model with fasting blood glucose levels comparable to those of control ON-DR mice [80]. Such a pre-diabetic animal model may be useful in examining whether the phenotype is a cause of hyperglycemia or a consequence of glucotoxicity. ON mice may also provide valuable information on the etiologies of diet-induced diabetes as an example of “gene-environment interaction” for the development of T2D in the modern obesogenic environment. In this point of view, the body weight phenotype of the two models (GK rats and ON mice) in comparison with their corresponding control strains is of great interest. For instance, insulin resistance is generally considered as a phenotype associated with obesity. However, the presence of insulin resistance in lean GK rats is inconsistent with this general principle and suggests that insulin resistance may be regulated, at least in part, independent of obesity at the genetic level. In contrast, the development of insulin resistance in HFD-fed ON-DP mice is consistent with the general principle, i.e., accompanying obesity. The obese ON-DP mice showed greater visceral fat accumulation, higher gene expression levels of inflammatory cytokines (Ccl2 and Tnf) in the visceral fat, higher blood pressure, and lower HDL-cholesterol levels compared to ON-DR mice [83]. The clustering of metabolic abnormalities indicates that ON-DP mice would be a suitable animal model of metabolic syndrome in humans.

Table 1 Phenotypic comparisons of GK rats and ON mice

		GK rats	ON-DP mice
Background strain		Wistar	C57BL6 × AKR × C3H
Breeding		Inbreeding*	Closed colony breeding
Selection index		Spontaneous hyperglycemia	High fat diet-induced hyperglycemia
Control strain	Outbred	Wistar	ON-DR
	Inbred	BN or Fisher	N/A
Pathogenesis		Polygene	Polygene × Environment (diet)
Diabetes onset		After weaning	After high fat diet
Hyperglycemia		Severe	Mild – Moderate
Obesity		Lean	Mild – Moderate
Insulin resistance		+	+
Islet shape		Starfish shape	Normal round shape
β-Cell mass		N/C or ↓	↑
β-Cell function		↓↓	↓
Glucose transport		↓	↓
ATP production		N/C or ↓	?
Ion channel activities		N/C	?
Exocytosis		↓	↓
Amplification by cAMP		↑ or ↓	?
Complications		Vascular, Heart, Eye, Kidney, Nerve	Atherosclerosis

Phenotypic comparisons of Goto-Kakizaki (GK) rats and Oikawa-Nagao Diabetes-Prone/Diabetes-Resistant (ON-DP/DR) mice. All comparisons were made at the onset of diabetes by the differences between each animal model and its corresponding control stain. *Genetic diversity has been observed among the colonies. BN, Brown Norway. N/A, Not applied. N/C, No change between the animal model and the control.

A common feature of the two rodent models is a defective stimulus-secretion coupling in pancreatic β-cells. Importantly, no causal relationship between the secretory dysfunction and the β-cell mass are identified in these models. Impaired insulin response in T2D has partly been considered as a consequence of decreased β-cell mass (quantitative deficit) [92]. On the other hand, impaired insulin secretion in T2D is more likely explained by defective β-cell function (qualitative defect) [92]. We recently characterized human islets of obese donors with either established T2D or being non-T2D (ND) [84]. Among the obese donors, islets from T2D individuals showed reduced GSIS in the perifusion system (an ex-vivo model for monitoring dynamic insulin release from pancreatic islets), while the basal insulin secretion level was similar to that of islets from obese ND. A static incubation with high K⁺ also showed reduced insulin secretion (KSIS) in the islets from obese T2D individuals, suggesting that defects exist downstream of the K_ATP channels in the β-cell stimulus secretion coupling. Indeed, capacitance recordings on single β-cells using the patch-clamp technique revealed that insulin granule exocytosis in β-cells from obese T2D was reduced by ~50% compared to those from obese ND. Genetic association studies have reported that most of the variants associated with T2D risk are related to β-cell function [93]. Our previous study using human β-cells actually demonstrated that the number of genetic variants in major diabetes susceptibility genes (in or near KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2) were associated with impaired insulin granule docking and exocytosis [94]. These findings indicate that the genetic background primarily regulates β-cell insulin secretion capacity, and that the functional impairments may precede the β-cell loss. Further studies on β-cells from the selectively bred animal models may provide novel insight into the etiology of T2D in humans.

Conclusion

By summarizing the background and phenotype of GK rats and ON mice, we highlight the potential advantages of the selectively bred rodent models for studying the etiology of T2D. Here, we suggest that GK rats and ON mice are useful animal models for understanding the mechanisms of impaired insulin secretion, particularly dysfunctions in the stimulus secretion coupling in β-cells. In the regulation of insulin secretion, calcium-dependent exocytosis is a promising target as signs of defective exocytosis are commonly observed in β-cells of human T2D donors, GK rats and ON mice. The selectively bred diabetes rodent models also have potential usefulness in the study of insulin resistance. Notable examples include systemic insulin resistance independent from obesity in GK rats and lower insulin-induced leptin production capacity in ON-DP mice. In the metabolic features associated with the development of diabetes, not only genetic risk variant like single nucleotide polymorphism but also epigenetic factors including DNA methylation and miRNA can be involved. The polygenic background of selectively bred rodent models polygenic background would be a potential advantage in the genetic and epigenetic studies on the etiology of T2D. Finally, it should be noted that the onset of T2D is largely influenced by adverse environmental factors including modern high-fat energy-dense diet. Thus, the unique feature of ON mice that mimic the gene-environment interactions in the development of human diabetes will become more meaningful in deciphering the complex etiology of T2D in the modern obesogenic environment.

Acknowledgment

All animal experiments were performed in accordance with ethical permits issued by the Malmö/Lund Ethical Committee of Animal Research (Malmö and Lund, Sweden) or the Nippon Medical School Animal Policy and Welfare Committee (Tokyo, Japan).

We thank Britt-Marie Nilsson, Anna-Maria Veljanovska Ramsay and Neelanjan Vishnu (Lund University) for technical assistance of GK/LU rat studies, Holger Luthman (Lund University) for valuable discussion regarding GK/LU rats, and Momoyo Kawahara (Nippon Medical School), Miki Onodera and Tomoo Nakamura (Institute for Animal Reproduction) for technical assistance of ON mice studies.

The work is financially supported by Japan Society for the Promotion of Science (MN and AA, JSPS KAKENHI Grant Numbers 25860300, 26460497, 15K08434, 17KK0184, 19K23872, 21K05453 and 22K07008), European Foundation for the Study of Diabetes and Japan Diabetes Society (MN), Uehara Memorial Foundation (MN), Scandinavia-Japan Sasakawa Foundation (MN), Sumitomo Life Welfare Foundation (MN), Ono Medical Research Foundation (MN), Diabetes Wellness Sverige (MN, 720-2964 JDWG), Lotte Shigemitsu Prize (AA), Swedish Foundation for Strategic Research (IRC-LUDC; Dnr IRC15-0067), Swedish Research Council (SFO-EXODIAB; Dnr 2009-1039), Swedish Research Council (LE, 2019-01406), Region Skåne-ALF (LE) and Swedish Diabetes Foundation (LE; DIA2019-454).

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